African trypanosomiasis is lethal if not treated and its incidence is increasing. Existing therapies are antique by contemporary standards and no vaccine is available. The DNA topoisomerases are enzymes essential for nucleic acid biosynthesis and cell survival and are proven targets for clinically valuable anti-infective and antitumor drugs. Previous work from this lab has established that topoisomerase inhibitors cause dramatic lesions in nuclear and mitochondrial (kinetoplast) DNA which are directly proportional to cell killing. This is an ongoing project to examine the DNA topoisomerases as targets for antitrypanosomal drug development, and the specific aims are three. First is to study type I enzymes, including the intracellular localization of structurally unique topo IB. This aim also includes the newly identified topoisomerases IA, characterizing their gene sequence and investigating intrace!!ular enzyme function by RNAi silencing. Aim 2 focuses on a previously unrecognized, second type IIA topoisomerase that occurs in trypanosomes: its catalytic features and inhibitor susceptibilities, and the consequences of silencing by RNAi. The third aim is to bring these basic molecular studies closer to the clinic by exploring the structure-activity relationship and selective toxicity of known topoisomerase inhibitors against trypanosomes in vitro. Included are recently discovered inhibitors oftopo IB, as well as topo II inhibitors with representatives currently in clinical trials. Compounds that appear most promising will be evaluated in mice. These studies take a multi-faceted, rational, and tangible approach to the development of much-needed new anti-trypanosomal chemotherapy.